ABSTRACT Tetrastigma hemsleyanum Diels et Gilg ( T. hemsleyanum ) is a plant of considerable medicinal and economic value. However, the molecular mechanisms underlying its tuberous root formation remain poorly understood. To investigate the molecular basis of tuberous root formation, we analyzed hormonal metabolic levels, transcriptomic profiles, and root anatomical changes during this process. Using ultra‐performance liquid chromatography‐electrospray ionization tandem mass spectrometry, we quantitatively assessed the levels of eight plant hormones and their derivatives in the early stages of tuberous root formation and in adventitious roots. The results revealed significant fluctuations in hormone levels, with a marked upregulation of cytokinins (tZ, DZ, and IP) and the complete absence of gibberellin GA 1 post‐tuberous root formation. Jasmonic acid content decreased, while methyl jasmonate (MeJA) increased substantially. Exogenous application of MeJA further confirmed the role of the jasmonic acid pathway in tuberous root formation, underscoring the pivotal role of these hormones in root differentiation and expansion. Additionally, transcriptomic analysis identified significant alterations in biological processes associated with the cytoskeleton and cell wall during tuberous root formation. Anatomical observations indicated reduced lignification and a notable increase in vascular cambium and xylem parenchyma cells. In conclusion, this study provides valuable insights into the molecular mechanisms of tuberous root formation in T. hemsleyanum , emphasizing the critical role of plant hormones and offering new strategies for enhancing tuber growth and yield through hormonal regulation.